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PubMed1995

Hypervariable genomic variation to reconstruct the natural history of populations: lessons from the big cats.

The extent and nature of variation in hypervariable regions DNA have been used in the past as a means to infer the natural histories of populations. We review the interpretation of the extent of genetic diversity for minisatellite DNA in the cheetah to estimate the timing of a population bottleneck in the species and the potential application of a second class of hypervariable DNA, microsatellite DNA, as a molecular tool to examine the natural histories of felid populations. A calibration curve relating the degree of allele fragment sharing in individuals to relatedness in a captive pedigree of cheetahs is presented. This measurement has important applications for management of potential matings in captive management situations.

Acinonyx↗
PubMed2000

Disulfide bonds in big ET-1 are essential for the specific cleavage at the Trp(21)-Val(22) bond by soluble endothelin converting enzyme-1 from baculovirus/insect cells.

Endothelin converting enzyme-1 (ECE-1) is a type II integral membrane protein and a zinc metalloendopeptidase. ECE-1 generates endothelin-1 (ET-1), the most potent vasoconstrictor yet discovered, by specific proteolytic processing of a precursor peptide, big ET-1. An insect cell expression system, which generates up to 4.3 mg of a secreted, soluble form of ECE-1 (solECE-1) per liter culture medium, has been established and solECE-1 was purified to homogeneity using five chromatographic steps. SolECE-1 expressed in insect cells could be suitable for X-ray structure determination as it is much less glycosylated than solECE-1 from mammalian cells. SolECE-1 from both sources, nonetheless, has comparable enzymatic properties. Despite apparent structural similarities, ECE-1 cleaves big ET-1 exclusively between Trp(21) and Val(22), in contrast to neprilysin, which cleaves big ET-1 at various sites. However, when linear big ET-1, in which the formation of disulfide bonds has been prevented by alkylation of the four cysteines, was used as substrate, it was cleaved by solECE-1 at multiple sites. This result indicates that secondary/tertiary structure of big ET-1 induced by disulfide bonds is essential for the specific cleavage of the Trp(21)-Val(22) bond by ECE-1. A continuous, fluorescent ECE-1 assay has been developed using a novel substrate, 2-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-(p-nitro-Phe(8))-Arg. This simple and rapid assay can greatly facilitate discovery of novel ECE inhibitors useful as pharmaceutical agents.

Amino Acid Sequence↗
PubMed2001

An electrophysiological study of neural pathways for corticofugally inhibited neurons in the central nucleus of the inferior colliculus of the big brown bat, Eptesicus fuscus.

This electrophysiological study tests the hypothesis that one possible neural pathway for corticofugally inhibited neurons in the central nucleus of the inferior colliculus (ICc) of the big brown bat, Eptesicus fuscus, is mediated through excitatory projections from the auditory cortex (AC) to the external nucleus of the IC (ICx), which then sends inhibitory inputs to the ICc. This study shows that all neurons in the ICx are broadly tuned to stimulus frequency. Electrical stimulation in the AC typically increases the number of impulses, expands the auditory spatial response areas, and broadens the frequency tuning curves (FTCs) of neurons in the ICx. This corticofugal facilitation is mediated at least in part through NMDA receptors, since application of DL-2-amino-5-phosphonovaleric acid (APV), an antagonist for NMDA, decreases these response properties of neurons in the ICx. Electrical stimulation in the ICx typically decreases the number of impulses, reduces the auditory spatial response areas, and narrows the FTCs of neurons in the ICc. This inhibition is mediated at least in part through GABAA receptors, since application of bicuculline, an antagonist for GABA, increases these response properties of neurons in the ICc. These data suggest that corticofugal facilitation of the ICx and the inhibition of the ICx to the ICc may be one of the polysynaptic pathways for corticofugal inhibition of neurons in the ICc. Possible functions of this polysynaptic pathway in acoustic orientation and signal processing are discussed.

Animals↗
PubMed1997

Orienteering competition injuries: injuries incurred in the Finnish Jukola and Venla relay competitions.

OBJECTIVES: The aim of this study was to find out the amount, type, and localisation of acute orienteering injuries in the 1995 Finnish Jukola (men's) and Venla (women's) relay competitions with 9724 participants. METHODS: Patient data were collected on a preplanned form at the first aid station. RESULTS: There were 244 acute competition injuries. The overall injury incidence was 2.5% (95% confidence interval (CI) 2.2 to 2.8), 2.3% in the men's relay and 3.2% in the women's relay, and the difference between incidences among men and women was statistically significant (odds ratio (OR) 1.4; 95% CI 1.1 to 1.9). The injury rates were also calculated per 1000 competition hours and per 1000 km and were 15.4 and 2.6 respectively. Most of the injuries could be treated at the first aid station. Female orienteers had more blisters (OR 4.4; 95% CI 2.5 to 7.9) and fewer wounds (OR 0.5; 95% CI 0.2 to 1.0) than male orienteers. Injuries occurred mostly in the lower extremities (70%), and ankle sprains accounted for 25% of all injuries. Injuries were more common during the first leg of both relays. CONCLUSIONS: This study shows the heavy workload in a first aid station of a big orienteering relay competition. It indicates a difference in injuries between the sexes and in the different stages of the relay, which should be further investigated in order to prevent orienteering competition injuries.

Ankle Injuries↗
PubMed2005

Investigating DNA adduct-targeted mutagenicity of tamoxifen: preferential formation of tamoxifen-DNA adducts in the human p53 gene in SV40 immortalized hepatocytes but not endometrial carcinoma cells.

Tamoxifen is a widely used drug for chemotherapy and chemoprevention of breast cancer worldwide. Tamoxifen therapy is, however, associated with an increased incidence of endometrial cancer. The carcinogenicity of tamoxifen is ascribed to its genotoxic and estrogen agonist effects. We investigated DNA adduct-targeted mutagenicity of tamoxifen as a function of its genotoxicity in the cII transgene in Big Blue mouse embryonic fibroblasts and mapped the formation of tamoxifen-induced DNA adducts in the p53 tumor suppressor gene in SV40 immortalized human hepatocytes and human endometrial carcinoma cells. We used the terminal transferase-dependent polymerase chain reaction for mapping of DNA adducts in the cII and p53 genes. We utilized a lambda phage-based assay and DNA sequencing for determining cII mutant frequency and mutation spectrum, respectively. Tamoxifen treatment yielded polymerase-blocking DNA adducts at multiple nucleotide positions along the cII transgene. The treatment significantly and dose-dependently increased the cII mutant frequency (p < 0.01), leaving a unique mutation spectrum (p < 0.0001) and a signature mutation of G:C --> T:A transversions (p < 0.03), relative to the control. Tamoxifen treatment of the immortalized human hepatocytes but not endometrial carcinoma cells, even in the presence of an external activation system, i.e., rat liver S9 mix, induced DNA adducts at specific codons along exons 6 and 8 of the p53 gene. These data suggest a proficient metabolic activation of tamoxifen in human liver and an inefficient activation and/or efficient detoxification of tamoxifen in human endometrium. Because the liver is essentially a mitotically quiescent organ, tamoxifen-DNA adduction in the liver may, at least partially, prevent its reactants from reaching highly proliferative organs via, e.g., circulating blood. Thus, tamoxifen-DNA adduction in the liver may not have as significant biological consequences as it might have in highly proliferative organs. Our findings favor an involvement of a nongenotoxic mechanism in tamoxifen-associated human endometrial cancer.

Animals↗
PubMedDate not supplied

Differences in adhesion of urothelial cells to a panel of substrates are characteristic for established human urothelial cell lines differing in their transformation grade.

Eight human urothelial cell lines established and propagated in the Fibiger Institute, differing in their transformation grade as described by Christensen et al were brought into suspension and contacted with a panel of seven substrates, ie. Con. A, Fibronectin, WGA, Collagen IV, Laminin, PNA and artificial basal membrane. Cell adhesion was measured on big cell populations with the use of flow cytophotometry, measured samples contained nonadhering cells and fluorescent beads, added as an internal standard. It was found that the cell lines differed from each other by the kinetics of cell adhesion to the chosen panel. A higher percentage of cells belonging to established cell lines adhered to PNA, WGA or Con A coated surfaces than to laminin, fibronectin or collagen IV. The difference in adherence of cells to fibronectin, laminin or collagen IV coated surfaces makes differentiation between the single cell lines possible. Discriminant analysis of the total patterns of adhesion of all cell lines was used for comparison. The effect of trypsinization on the structure of the cell surface, accompanied by possible changes in adherence, was evaluated by comparison of the immediate results with those obtained with cells after 3 hours of recovery at 37 degrees C. It was found that all cell lines showed a better adherence to laminin after recovery. This is consistent with the published data on the high sensitivity of laminin receptors to trypsinization. On the other hand, the receptors for PNA, WGA and Con A were less expressed after the recovery period, as evidenced by a decreased adherence of the cells to these three substrates. The effect of recovery of cells on the amount of their receptors for fibronectin and collagen IV was different in different cell lines, but the direction of change for this pair of receptors was identical in a given cell line. In some instances the recovery period had no effect whatsoever on cell adherence. The different pattern of adhesion of cells belonging to established cell lines to the chosen panel of substrates can be used as a parameter for characterization of single cell lines, but it does not correlate with their grade of transformation.

Cell Adhesion↗
PubMed1994

High yield expression and purification of human endothelin-1.

A DNA construct encoding human big endothelin (Big ET) preceded by the factor Xa protease recognition site (Ile-Glu-Gly-Arg), fused in frame to the maltose binding protein sequence, has been introduced in DH5-alpha cells. The fusion product (MBP-Big ET) was expressed at a concentration close to 100 micrograms/ml of culture broth and constituted approximately 50% of the total protein content. Crude cell extracts containing the fusion product have been directly treated with trypsin under mild denaturing conditions in order to release big endothelin (1-37) from the adduct. Cleavage yield of the MBP-Big ET adduct was close to 70%. Big ET(1-37) was separated from unrelated peptides derived from the tryptic digest of the bacterial extract by affinity chromatography. The affinity column was prepared by immobilizing a protease resistant peptide ligand able to recognize Big ET with sufficient affinity, selectivity, and specificity. From the affinity step (recovery, 90%), recombinant Big ET(1-37) was obtained with a purity close to 80%. The affinity-purified recombinant product was then digested with alpha-chymotrypsin in order to release endothelin (1-21), which was then purified by RP-HPLC. With this two-step purification protocol, 3 micrograms of endothelin was recovered from 1 ml of bacterial broth, with a purity close to 95%.

ATP-Binding Cassette Transporters↗
PubMed1992

A rabbit antiserum raised against the hexapeptide endothelin(16-21) shows different binding affinities for endothelin-1, -2 and -3.

A antiserum raised against the C-terminal hexapeptide ET16-21 common to ET-1, -2 and -3 was produced and characterized with respect to its binding properties for ET-1, -2, -3, ET16-21, the C-terminal octapeptide ET14-21, its derivative Phe21-ET14-21 and human big-ET-1. The antibody reacted with the peptides with decreasing binding affinities in the order: ET-1 greater than ET-2 greater than or equal to ET16-21 = ET 14-21 much greater than Phe21-ET14-21. It showed no crossreactivity with human big-ET-1. Similar results were obtained using [125I]ET-1, -2 or -3 as tracer. Substitution of Trp21 by Phe decreased the binding affinity of ET14-21 about 10 fold. Thus, the immunologically recognized sequence of the peptides is C-terminal and Trp21 seems to be important for high binding affinities. The significant differences in binding affinity observed for ET-1, -2, -3 and ET16-21 are consistent with an interaction of the C-terminal part of the endothelins with the bicyclic N-terminal part.

Amino Acid Sequence↗
PubMed2026

Differential Mutagenic Response of Rat Liver and Lung to Nicotine-Derived Nitrosamine Ketone (NNK).

Nitrosamines (NA) are chemical impurities that are present in tobacco, foods, more recently in some pharmaceuticals and are associated with genotoxicity and carcinogenicity. We evaluated the in vivo mutagenicity of nicotine-derived nitrosamine ketone (NNK) or 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone, a model compound used as an anchor molecule to estimate carcinogenic potency of unknown nitrosamine impurities. Big Blue rats were treated with NNK at doses ranging from 0.001 to 30 mg/kg for 28 days, following which liver and lung tissue were harvested 3 days later for nuclear genomic DNA isolation. Mutations in liver and lung were assessed with the cII transgene assay and endogenous genomic loci using Duplex Sequencing (DupSeq), a highly validated error-corrected sequencing (ECS) technology. The no genotoxic effect level (NOGEL) was 1 mg/kg in liver and 0.1 mg/kg in lung while the benchmark dose (BMD) analysis for cII mutagenicity determined a BMDL50 of 1.3 mg/kg in liver and 0.12 mg/kg in lung, consistent with lung being the more sensitive target organ for carcinogenicity for NNK. ECS-derived mutagenicity was highly correlated with cII-derived mutagenicity. Interestingly, the types of mutations formed appeared to be tissue-specific with higher C > T transitions and lower T > G transversions in lung compared to liver, differences that may reflect tissue-specific DNA repair capacity and/or metabolic differences. Collectively, these data support the use of in vivo mutagenicity data&#x2500;from both TGR cII and ECS methods&#x2500;for human health and cancer risk characterization of nitrosamines and for estimating acceptable daily intakes for unknown nitrosamine drug substance related impurities.

Animals↗
PubMed1991

Heterologous in vivo processing of human preproendothelin 1 into bioactive peptides.

Endothelin (ET) is an extremely potent vasoconstrictor peptide of 21 amino acids, originally found in the supernatant of cultured vascular endothelial cells. To gain insights into its biosynthetic pathway, we expressed a synthetic RNA coding for the 212-amino acid precursor of human ET-1 (preproET-1) in Xenopus oocytes. Cell homogenates and oocyte incubation medium were tested by RIA using an anti-ET-1 serum. ET-1-like immunoreactivity was detected in oocytes injected with preproET-1 synthetic RNA but not in control oocytes and was much higher in medium than in cell homogenates. When preproET-1 was expressed in oocytes treated with monensin, a dramatic decrease in secretion of immunoreactive material was observed, indicating that secretion is mediated by the Golgi complex. ET-1-like immunoreactive material present in oocyte incubation medium was fractionated by reverse-phase HPLC into two main peaks, corresponding to the retention times of human big ET-1 and ET-1. Incubation medium of oocytes expressing the synthetic preproET-1 RNA elicited a characteristic vasoconstrictor response on rabbit vena cava, consistent with the biological activity that would be predicted from the amount of ET-1-like immunoreactivity measured. These results suggest that common pathways of ET maturation exist in widely different cells and that Xenopus oocytes may represent a useful tool in studying the cell biology of ET-1 synthesis.

Animals↗
PubMed2004

Primary structure of dog preproendothelin-3 and elevated gene expression in kidney affected with interstitial nephritis.

To compare the structure of the precursor polypeptide of dog endothelin-3, preproendothelin-3 (PPET-3), with the PPET-3 of other mammals, we cloned dog cDNA from lung tissue using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. An open reading frame encoding a 198-amino-acid polypeptide was found in the cDNA. Regions corresponding to a bioactive mature endothelin-3 peptide, an intermediate form known as big-endothelin-3 and an endothelin-3- like peptide were observed in the putative PPET-3. Comparative analysis showed that the similarity of the dog open reading frame sequence with those from human hypothalamus, mouse intestine, and rat eye is 76.2%, 69.5% and 66.3%, respectively, and that the similarity at the amino acid level is 65.6%, 59.8% and 58.8%, respectively. RT-PCR demonstrated significant elevated expression of PPET-3 mRNA in the kidney of dog with interstitial nephritis.

Amino Acid Sequence↗
PubMed2002

The putative capsid protein of the newly identified avian hepatitis E virus shares antigenic epitopes with that of swine and human hepatitis E viruses and chicken big liver and spleen disease virus.

We recently identified a novel virus, designated avian hepatitis E virus (avian HEV), from chickens with hepatitis-splenomegaly (HS) syndrome in the USA. We showed that avian HEV is genetically related to swine and human HEVs. Here we report the antigenic cross-reactivity of the putative open reading frame 2 (ORF2) capsid protein of avian HEV with those of swine and human HEVs and the Australian chicken big liver and spleen disease virus (BLSV). The region encoding the C-terminal 268 amino acid residues of avian HEV ORF2 was cloned into expression vector pRSET-C. The truncated ORF2 protein was expressed in E. coli as a fusion protein and purified by affinity chromatography. Western blot analysis revealed that the avian HEV ORF2 protein reacted with antisera against the Sar-55 strain of human HEV and with convalescent antisera against swine HEV and the US2 strain of human HEV, as well as with antiserum against BLSV. Convalescent sera from specific-pathogen-free chickens experimentally infected with avian HEV also reacted with the recombinant capsid proteins of swine HEV and Sar-55 human HEV. Antisera against the US2 human HEV also reacted with recombinant ORF2 proteins of both swine HEV and Sar-55 human HEV. The antigenic cross-reactivity of the avian HEV putative capsid protein with those of swine and human HEVs was further confirmed, for the most part, by ELISA assays. The data indicate that avian HEV shares certain antigenic epitopes in its putative capsid protein with swine and human HEVs, as well as with BLSV. The results have implications for HEV diagnosis and taxonomy.

Amino Acid Sequence↗
PubMed2000

A miniaturised semiautomated system for the identification of Yersinia species within the genus Yersinia.

Commercially available identification systems based on biochemical reactions of bacteria are not suited for typing the species of the genus Yersinia (Y.) or the biovars (BV) of the species Y. enterocolitica. This failure is caused by the limited number of biochemical reactions applied, resulting in the absence of important discriminatory key reactions. The MICRONAUT identification system (Merlin, Bornheim-Hersel) makes use of dried substrates/enzymes reactions in the wells of a 96-well microtitration plate, reading of the results by a scanner device and typing of the isolate by the calculation of probabilities according to a data base. For this study a special identification panel was designed on which 38 substrates and enzyme reactions were configurated including 20 reactions for the identification of the species of the genus and the Y. enterocolitica biovars. The database was calculated using the results obtained from a total of 250 Yersinia strains of the eleven species of the genus. Reevaluation of the results of these strains revealed an overall sensitivity of 98%, as only four strains were not identified satisfactorily. Considering also questionable results the sensitivity was still 85%. The system was also used to identify Y. pestis isolates, but in this case reading was done visually. The printouts usually cite species designation, identification quality and probabilities. The sealing of the plates in an aluminium bag guarantees long life and long lasting quality. However, an evaluation of the system with a considerable number of strains has to be done in a next step. The 'Yersinia identification set' can replace time-consuming tube testing in the future and is a big step forward towards a sensitive identification of Yersinia isolates in the routine laboratory.

Automation↗
PubMed1979

[Calcium requirement in infants with intrauterine dystrophy. Report I. Indicators of calcium balance in infants with intrauterine dystrophy].

Observations of many investigators point at a lower amount of calcium in body composition of Small-for-Date infants (S-f-D). So far, only few investigations indicated that the calcium metabolism is disturbed in the postnatal life of S-f-D infants as well. Taking into consideration these data, the authors attempt to define calcium-requirement in S-f-D infants. In this report the cross-sectional observations of 30 infants are described . They were born after 37 weeks of gestation. The mean body weight was 2318 and it ranged between 1200-2760 g. Their body weight at birth was below 10 percentile according to Brzozowska . In these infants 3-days balance studies were carried out once a month. The calcium balance indices were compared with the indices of the Full-Size infants (FS) who were observed during the former years. The S-f-D and FS infants were fed similar diets based on unmodified cow's milk formulas. It was noted that the percentage absorption index in S-f-D infants was significantly lower than in the control group in 1-st and 2-nd trimester of life (table III, fig. 2). The S-f-D infants had a good appetite in first semester of life and they ate a big volume of milk formula and calcium in it. It seems that this is the reason that calcium retention in S-f-D infants in spite of lower ability of calcium retention, did not differ statistically from calcium retention in FS infants. The mean values were a little lower however. In the 2-nd half year of life statistically significant difference between any of the balance indices in S-f-D and FS infants was not found.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed2006

Pioglitazone for type 2 diabetes mellitus.

BACKGROUND: Diabetes has long been recognised as a strong, independent risk factor for cardiovascular disease, a problem which accounts for approximately 70% of all mortality in people with diabetes. Prospective studies show that compared to their non-diabetic counterparts, the relative risk of cardiovascular mortality for men with diabetes is two to three and for women with diabetes is three to four. The two biggest trials in type 2 diabetes, the United Kingdom Prospective Diabetes Study (UKPDS) and the University Group Diabetes Program (UGDP) study did not reveal a reduction of cardiovascular endpoints through improved metabolic control. Theoretical benefits of the newer peroxisome proliferator activated receptor gamma (PPAR-gamma) activators like pioglitazone on endothelial function and cardiovascular risk factors might result in fewer macrovascular disease events in people with type 2 diabetes mellitus. OBJECTIVES: To assess the effects of pioglitazone in the treatment of type 2 diabetes. SEARCH STRATEGY: Studies were obtained from computerised searches of MEDLINE, EMBASE and The Cochrane Library. The last search was conducted in August 2006. SELECTION CRITERIA: Studies were included if they were randomised controlled trials in adult people with type 2 diabetes mellitus and had a trial duration of at least 24 weeks. DATA COLLECTION AND ANALYSIS: Two authors independently assessed trial quality and extracted data. Pooling of studies by means of random-effects meta-analysis could be performed for adverse events only. MAIN RESULTS: Twenty-two trials which randomised approximately 6200 people to pioglitazone treatment were identified. Longest duration of therapy was 34.5 months. Published studies of at least 24 weeks pioglitazone treatment in people with type 2 diabetes mellitus did not provide convincing evidence that patient-oriented outcomes like mortality, morbidity, adverse effects, costs and health-related quality of life are positively influenced by this compound. Metabolic control measured by glycosylated haemoglobin A1c (HbA1c) as a surrogate endpoint did not demonstrate clinically relevant differences to other oral antidiabetic drugs. Occurrence of oedema was significantly raised. The results of the single trial with relevant clinical endpoints (Prospective Pioglitazone Clinical Trial In Macrovascular Events--PROactive study) have to be regarded as hypothesis-generating and need confirmation. AUTHORS' CONCLUSIONS: Until new evidence becomes available, the benefit-risk ratio of pioglitazone remains unclear. Different therapeutic indications for pioglitazone of the two big U.S. and European drug agencies should be clarified to reduce uncertainties amongst patients and physicians.

Diabetes Mellitus, Type 2↗
PubMed2005

The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf.

BACKGROUND: Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs) that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. RESULTS: Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7). CONCLUSION: Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.

ADP-Ribosylation Factors↗
PubMed2006

Institutional conditions and individual experiences in the career-entry period of Swiss medical residents--a qualitative study.

BACKGROUND AND OBJECTIVES: The study investigated first-year residents' career entry experiences according to gender, clinical field and type of training hospital. In addition to quantitatively assessed workplace experiences, this paper reports qualitative data on institutional conditions as well as interpersonal and individual experiences encountered by junior physicians during their first year of residency. METHODS: The present study is part of the longitudinal Swiss physicians' career development study. After their first year of residency, participants were interviewed for the second time. The qualitative data of this second assessment are reported in this paper. A total of 1861 terms were given by 342 junior physicians working in the two main clinical fields (internal medicine and surgical fields) with regard to positive and negative experiences in the career entry period. The answers were assigned to 12 categories (according to Mayring's content analysis). These categories were then allocated to three superordinate subject areas: Institutional conditions, Interpersonal experiences, and Individual experiences. RESULTS: Institutional conditions accounted for 17% of all entries, with negative experiences in particular, high workload - predominating. Within Interpersonal workplace experiences (41% of all responses), relationship experiences accounted for the largest proportion of all statements (25.7%), which were mainly positive. Individual experiences (42%) were assessed both positively (professional competence, learning, responsibility, and pleasure) and negatively (working under pressure and curtailment of one's private life). Female and male residents reported similar workplace experiences. Residents in surgical fields more often complained of a lack of professional support than those in internal medicine. Physicians working in university or big county hospitals had more negative experiences with regard to teaching than residents at smaller hospitals. CONCLUSION: The junior physicians' career-entry experiences indicated that a genuine concept of education and training would greatly improve their workplace experiences. Furthermore, senior physicians should bear in mind that they are important role models for the junior staff.

Adult↗
PubMed1998

Methylation of CpG dinucleotides in the lacI gene of the Big Blue transgenic mouse.

Cytosine residues at CpG dinucleotides can be methylated by endogenous methyltransferases in mammalian cells. The resulting 5-methylcytosine base may undergo spontaneous deamination to form thymine causing G/C to A/T transition mutations. Methylated CpGs also can form preferential targets for environmental mutagens and carcinogens. The Big Blue(R) transgenic mouse has been used to investigate tissue and organ specificity of mutations and to deduce mutational mechanisms in a mammal in vivo. The transgenic mouse contains approximately 40 concatenated lambda-like shuttle vectors, each of which contains one copy of an Escherichia coli lacI gene as a mutational target. lacI mutations in lambda transgenic mice are characterized by a high frequency of spontaneous mutations targeted to CpG dinucleotides suggesting an important contribution from methylation-mediated events. To study the methylation status of CpGs in the lacI gene, we have mapped the distribution of 5-methylcytosines along the DNA-binding domain and flanking sequences of the lacI gene of transgenic mice. We analyzed genomic DNA from various tissues including thymus, liver, testis, and DNA derived from two thymic lymphomas. The mouse genomic DNAs and methylated and unmethylated control DNAs were chemically cleaved, then the positions of 5-methylcytosines were mapped by ligation-mediated PCR which can be used to distinguish methylated from unmethylated cytosines. Our data show that most CpG dinucleotides in the DNA binding domain of the lacI gene are methylated to a high extent (>98%) in all tissues tested; only a few sites are partially (70-90%) methylated. We conclude that tissue-specific methylation is unlikely to contribute significantly to tissue-specific mutational patterns, and that the occurrence of common mutation sites at specific CpGs in the lacI gene is not related to selective methylation of only these sequences. The data confirm previous suggestions that the high frequency of CpG mutations in lacI transgenes is related to the presence of 5-methylcytosine bases.

Animals↗